Antibodies, and particularly IgG antibodies, are the basis of some of the most successful therapeutics developed over the last 20 years (e.g., bevacizumab, rituximab, infliximab, adalimumab, trastuzumab, or cetuximab, to name but a few). This success is at least in part attributable to the fact that they are highly specific, have long serum half-lives, and can be produced relatively routinely, making them ideal drugs for immunotherapy. The basic structure of an antibody molecule (or immunoglobulin, Ig) is comprised of two identical heavy and two identical light polypeptide chains. These chains are linked by disulfide bonds forming a “Y”-shaped structure. Human immunoglobulins can be categorized into five classes (IgG, IgA, IgD, IgE, and IgM) referencing the heavy chain. IgG and IgA antibodies are further separated into four (IgG1-4) and two subclasses (IgA1-2), respectively. Recognition of specific antigens is mediated by the antigen-binding fragment (Fab), which includes the variable regions and one constant domain of the light and heavy chains. Effector functions are initiated by binding of the fragment-crystallizable region (Fc), corresponding to the other two domains of the constant region of the heavy chain (CH2 and CH3), to effector proteins such as Fc receptors (FcRs). Thus, the Fab fragments are comprised of variable and constant domains of light and heavy chains, while Fc fragments are comprised entirely of constant domains of heavy chains. This Fc domain prolongs the serum half-life of antibodies due to pH-dependent binding to the neonatal Fc receptor (FcRn), which salvages the protein from being degraded in endosomes.
Given the long serum half-life of antibodies, construction of Fc-fusion proteins has been implemented to prolong the half-life of therapeutic proteins, as most biologically active proteins and peptides have very short serum half-lives due to fast renal clearance, which limits their exposure in the target tissue and, consequently, their pharmacological effects. The Fc-fusion strategy also met with considerable success: marketed Fc-fusion proteins include, e.g., etanercept, alefacept, abatacept, rilonacept, romiplostim, belatacept, and aflibercept. As an additional benefit, the Fc portion of Fc-fusion proteins allows easier expression and protein A-affinity purification, which confers practical advantages in the development of antibody and Fc-fusion therapeutics.
Antibody engineering approaches have been used to further advance the clinical success of therapeutic antibodies, e.g., by altering their binding properties to ligand or Fc receptors, or by further extending their half-life. Typical approaches to achieve this include introducing mutations or altering glycosylation of the antibodies. Introducing mutations in the Fc chain has the inherent drawback of no longer working with natural sequences. Contrary to glycosylation of therapeutic proteins, which is generally accepted to prolong circulating half-life, studies on the effect of glycosylation on the elimination rate of immunoglobulins from circulation have produced conflicting results (Millward et al., 2008), and most studies conclude that glycan structural differences of the Fc moiety do not affect clearance (Chen et al., 2009).
During post-translational modification of the antibody chains, enzymes in the endoplasmic reticulum and Golgi apparatus can attach carbohydrate chains to the polypeptide backbone of the antibody. A single N-linked glycan is present in the Fc portion of all IgG subclasses, at an asparagine at position 297 (Kabat numbering). About 20% of IgG antibodies contain glycans elsewhere on the molecule (Jefferis, 2005). Most recombinant antibody drugs have been engineered or selected to contain only the single Fc glycosylation site.
When the antibody chains are correctly folded and associated, the oligosaccharide at position 297 is sequestered within an internal space enclosed by the CH2 domains, and there are extensive non-covalent interactions between the oligosaccharide and the amino acids of antibody, resulting in reciprocal influences on conformation.
The oligosaccharides found at the conserved Asn-297 site are typically of a fucosylated biantennary complex type. However, among antibody molecules, there may be considerable heterogeneity in the carbohydrate structures (glycoforms) due to altered branching, chain length and/or altered number of carbohydrate moieties. Indeed, the structure of the attached N-linked oligosaccharides varies considerably, depending on the degree of processing, and can include high-mannose, as well as complex biantennary oligosaccharides with or without bisecting GlcNAc and core Fucose residues (Wright and Morrison, 1997). Typically, there is heterogeneous processing of the core oligosaccharide structures attached at a given glycosylation site, with the result that even monoclonal antibodies exist as multiple glycoforms. Moreover, major differences in antibody glycosylation occur between antibody-producing cell lines, and even minor differences are seen for a given cell line grown under different culture conditions.
Indeed, each step in mammalian N-glycan biosynthesis (FIG. 1A, top) is <100% efficient, and some enzymes compete for substrates, resulting in many different glycoforms. Heterogeneous glycosylation presents problems in the production of therapeutic proteins. For example, glycans can affect pharmacokinetics and biological activities (Ferrara et al., 2006; Elliott et al., 2004); however, N-glycans are often crucial for protein folding, so these difficulties cannot be overcome by completely removing N-glycosylation sites or interfering with glycosylation before or in the endoplasmic reticulum.
The differences in glycoforms may result in different or inconsistent effector functions, which can render the antibodies difficult to use therapeutically or define from a regulatory point of view. Also, glycoforms that are not commonly biosynthesized in humans may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. Deglycosylating the Fc moiety at position 297 can result in decreased or eliminated effector functions of the Fc-containing molecules, or in reduced stability (Krapp et al., 2003; Yamaguchi et al., 2006; Barb et al., 2011; Buck et al., 2013).
It would be advantageous to obtain Fc-containing molecules that have improved properties, such as longer circulating half-life, but without drawbacks such as heterogeneous glycosylation or reduced antigen binding.